TRP (transient receptor potential), CB (cannabinoid) and ASCI (acid-sensing) channels are thought to act together to sense, transmit, and integrate the pain response and the associated inflammatory responses. TRPV1, in particular, interacts with a multiple endogenous and exogenous molecules and stimuli, and is considered a key component in the integration of inflammatory pain. Project 3 will investigate relationships between bioactive Echinacea and Hypericum constituents and TRPV1 and other channels. It will test the hypotheses that Echinacea and Hypericum contain constituents with bioactivity against a range of TRPV1- related receptors, and that biochemical constituents of Echinacea induce pain-receptor responses by mechanisms distinct from those of capsaicin, a TRPV1 agonist. For the proposed studies, ion channel bioactivity will be assayed using transient-expression two models: 1) a frog oocyte system for initial screenings of multiple plant fractions, and 2) a human HEK293t cell model for more detailed evaluation of the role of the plant constituents in integration of inflammatory pain. Specific Aim 1: Evaluate the breadth of bioactivity of Echinacea and Hypericum on TRP, CB, and ASCI channels. These experiments will reveal whether these extracts activate channels other than TRPV1, and whether each activity is invoked by a distinct complement of botanical constituents. Specific Aim 2: Identify constituent(s) of Echinacea and Hypericum that evoke TRPV1-channel responses using bioactivity-guided fractionation. Extracts will be iteratively fractionated, tested for TRPV1 activation, and analytically evaluated for chemical composition. Our goal is to identify particular compounds and classes of plant compounds that induce TRPV1 bioactivity. Specific Aim 3: Identify mechanisms of TRPV1 bioactivity via Echinacea constituents. Inflammation and pain are complexly interrelated. In this aim, we seek to characterize how Echinacea extracts and constituents interact with other agonists and antagonists. Furthermore, transcriptomic analyses, in parallel with assays of anti- and pro- inflammatory responses, will reveal genetic and signaling networks affected by Echinacea constituents in a TVPR1-dependent manner. Taken together, these studies will address the cellular mechanisms by which these botanicals might influence TRPV1/-mediated integration of pain and inflammatory responses.